P1 and P2' site mutations convert protease nexin-2 from a factor XIa inhibitor to a plasmin inhibitor.

The kunitz protease inhibitor domain of PN2 (PN2KPI) is a potent and specific inhibitor (Ki 0.5–2 nM) of factor XIa (FXIa) and inhibits cerebrovascular thrombosis in mice. To determine whether the antithrombotic properties of PN2KPI arise from its FXIa-inhibitory activity, we have now prepared mutant forms of PN2KPI. Mutations at the P1 (Arg15) site in combination with P2′ (Met17) mutations profoundly affect inhibition of FXIa, plasmin, kallikrein, factor Xa and thrombin. The mutant proteins PN2KPI-R15K, -M17K, -R15K,M17K and -R15K,M17R lost inhibitory activity against FXIa (Ki 34, 94, 3081 and 707 nM, respectively) and kallikrein (no inhibition) and gained inhibitory activity against plasmin (Ki 108, 7, 8 and 8 nM, respectively). The intravenous administration of rPN2KPI into mice dramatically decreased thrombus formation in a murine model of FeCl3-induced carotid injury, whereas rPN2KPI-R15K,M17K failed to inhibit thrombus formation. Molecular modelling studies showed that fine structural variations explain the observed functional differences in FXIa and plasmin inhibition. PN2KPI has potent antithrombotic activity due to its specific FXIa anticoagulant activity, whereas PN2KPI-R15K,M17K and PN2KPI-R15K,M17R have potent antifibrinolytic (antiplasmin) activity without anticoagulant or antithrombotic activity.